1. Field of the Invention
The present invention relates to transgenic bioluminescent plants. More specifically, the present invention relates to plants that have been transfected via agro-bacterian or other means known to those in the art with genes encoding luciferase and luciferin such that they may glow in the dark. The transfected genes may be regulated by promoter regions designed to regulate the timing and duration of the genetically engineered bioluminescence.
2. Prior Art
It has been known in the art for some time that certain enzymes called luciferases will bioluminescence in the presence of their substrates, known as luciferins. Luciferases are a broad class of proteins that may be found in bacteria, jelly fish, fireflies and a variety of other organisms. Most luciferases share very little homology with one another and are thought to have evolved relatively independently of each other. Several luciferase genes have been identified and their bioluminescent activities have been used extensively to study gene regulation and expression. By inserting a luciferase protein downstream from a promoter to be studied, one may easily tell when that promoter has been activated by the resulting bioluminescence.
Luciferins tend to be complex organic molecules. Some are thought to be formed by means of complex catabolic pathways. Others, such as coelenterazine, result from the cyclization of amino acids in an expressed polypeptide. Until recently, it has not been possible to isolate a gene capable of forming luciferin in vivo. This has meant that in order to detect luciferase, luciferin must be directly applied to the organism expressing luciferase. Because the luciferin must be absorbed by the cells, individual cells and relatively thin tissue cultures, including very small seedlings, have been the only suitable hosts for studying gene expression using luciferase. Often, the organisms expressing luciferase are lysed and then exposed to a luciferin solution. This obviously kills the host organism.
There has been a significant amount of work done to improve the use of luciferase in studying gene expression. However, they have all been limited by the inability to produce in vivo bioluminescence without the addition of chemicals outside a laboratory environment and in larger organisms.
U.S. Pat. No. 5,093,240 to Inouye et al. discloses the transgenic use of aequorin and derivative polypeptides. This patent discloses the use of a luciferase enzyme in a vector designed for mass production. It is contemplated that the patent will be used to grow large quantities of luciferase in a bacterial culture. It does not disclose the addition of genes capable of forming intracellular luciferin. It also does not contemplate or disclose suitable methods for inserting luciferin into a plant cell.
U.S. Pat. No. 5,162,227 to Cormier also discloses recombinant DNA vectors into which a luciferase enzyme has been inserted. Like the above patent, it contemplates use of these vectors for mass production of luciferase in the bacterial culture. It contemplates use of the luciferase gene as a marker, or selection, gene sequence. It does not contemplate the addition of a luciferin gene into the vector, in vivo bioluminescence or the formation of a transfection vector suitable for plant cells.
U.S. Pat. No. 5,422,266 to Cormier et al. discloses an invention very similar to the one in the above paragraph. It discloses the insertion of a luciferase gene into a vector suitable for use in microorganisms. Like the above mentioned patent, it does not contemplate the additional insertion of a luciferin gene, in vivo bioluminscence or use of vectors suitable for insertion into plant cells.
U.S. Pat. No. 5,583,024 to McElroy et al. discloses use of another luciferase enzyme to be used in a transcription assay. The patent contemplates use of the luciferase to quantify transcription levels of various promoter sequences. It requires lyses, and thus death, of the transformed cells. It does not contemplate in vivo bioluminscence or the addition of a luciferin gene.
U.S. Pat. No. 5,976,796 to Szalay et al. discloses a fusion protein comprising a luciferase and a fluorescing protein. The patent contemplates its use as a double marker in transcription assays. It does not provide for intracellular luciferin or in vivo bioluminescence.
U.S. Pat. No. 5,221,623 to Legocki et al. discloses the use of the lux bacterial luciferase gene in transcription assays of various promoters. It does not contemplate in vivo bioluminsence in mature plants or the incorporation of a luciferin gene. Furthermore, the lux bioluminescence mechanism requires a substantial concentration of organic aldehydes. The patent discloses applying aldehyde vapors to the microorganisms. This would be impractical for use in the present invention.
U.S. Pat. Nos. 5,876,995 and 6,247,995 B1 both to Bryan disclose the use of bioluminescent luciferase/luciferin mechanisms for use in a wide variety of novelty items. The luciferase enzyme is added to a large variety of products and the luciferin is added subsequently. This patent does not disclose recombinant uses for luciferase recombinant DNA. However, the specification of this patent is very useful in that it gives a very detailed, textbook-like description of the entire field of bioluminescence.
U.S. Pat. No. 5,741,668 to Ward et al. discloses a polypeptide capable of spontaneously forming coelenterazine in vivo. This is the only patent found in the prior art that contemplates in vivo expression and production of a luciferin. As coelenterazine is difficult to harvest in red pansies and other organisms, this patent contemplates mass producing coelenterazine by expressing and harvesting it in bacterial cultures. It does not contemplate combining this gene with a luciferase gene in a single vector and using that vector to form bioluminescent mature plants.
It is therefore desirable to provide a method for causing bioluminescence in a mature multi cellular organism.
It is also desirable to provide a method for inducing bioluminescence without the need of applying chemicals to an organism.
It is also desirable to provide for a mature plant capable of bioluminescence outside of a laboratory setting and without the need of applying special chemicals.
It is also desirable to provide a mature plant capable of bioluminescence where the timing of that bioluminescence is controlled.